Overexpression of succinyl-CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in engineered Escherichia coli BL21(DE3).

AIM: This study aims to increase the 3-hydroxyvalerate (3HV) fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] using succinyl-CoA synthase. METHODS AND RESULTS: Escherichia coli YH090, a polyhydroxyalkonate (PHA)-producing strain, was further engineered for overexpression of succinyl-CoA synthase genes (sucCD), and examined for P(HB-co-HV) copolymer production in the presence of various precursor molecules using mixture analysis. Glycerol, succinate and propionate were screened as important factors for controlling intracellular PHA accumulation and monomer composition. Glycerol concentrations exerted the greatest influence on the overall biomass concentration and the intracellular PHA content, while propionate concentrations in the presence of succinate influenced the 3HV content of the copolymer. Mixture analysis also demonstrated that the engineered strain has the capacity to accumulate up to 80% of its cell dry weight (CDW) as PHA with a variable fraction of 3HV monomer (maximum of 72 wt %) depending on the controlled conditions. CONCLUSIONS: Propionate is the principal precursor for 3HV monomer in P(HB-co-HV) biopolymer and its utilization requires conversion to propionyl-CoA. Engineered E. coli YHY99, overexpressing sucCD genes, leads to an increase of the succinyl-CoA pool, which enhances the conversion rate of propionate by providing a CoA supply to other acyltransferase enzymes that have a role in propionate utilization. SIGNIFICANCE AND IMPACT OF THE STUDY: Engineered E. coli YHY99 was able to utilize propionate with a 4·5-fold increase in rate, as compared to the control strain, and resulted in the synthesis of a copolymer with high 3HV monomer content.

Inactivation of the lpcC gene alters surface-related properties and symbiotic capability of Bradyrhizobium japonicum.

UNLABELLED: We investigated the role of the Bradyrhizobium japonicum lpcC gene, encoding a mannosyl transferase, involved in the lipopolysaccharide (LPS) biosynthesis. The inactivation of the lpcC gene considerably altered the LPS structure and the cell surface properties. LPS analysis showed that the lpcC mutant JS715 had an abnormal LPS structure deficient in O-antigen. The cell surface hydrophobicity increased approximately threefold in JS715 compared to the wild type. The increased cell surface hydrophobicity is likely to be related with cell aggregation in the mutant culture. For the growth comparison, JS715 showed slower growth rate than the wild type. The motility of JS715 decreased in soft agar plates, but it showed enhanced biofilm-forming ability. Interestingly, JS715 was not able to nodulate the host legume soybean (Glycine max). This study shows not only that lpcC is involved in the biosynthesis of O-antigen in the B. japonicum LPS, but also that inactivation of the lpcC gene affects symbiotic capability of B. japonicum and surface-related properties such as cell hydrophobicity, biofilm formation and motility. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the role of the B. japonicum lpcC in nodulation with soybean and importance of cell surface hydrophobicity. The results also highlight that intact LPS is required for successful symbiosis between B. japonicum and soybeans. Our findings not only support previous studies emphasizing the necessity of LPS on the interaction between the two symbiotic partners, but also contribute to a better understanding of the symbiotic mechanisms.

Analysis of the association between air pollution and allergic diseases exposure from nearby sources of ambient air pollution within elementary school zones in four Korean cities.

The objectives of this study were to survey elementary school students regarding the environmental conditions of their elementary schools and to assess the relationship between air pollution and allergic disease using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. Therefore, this study was designed as a cross-sectional study. In this study, seven elementary schools were selected and they were classified into three categories. The selection included one school with no traffic-related or other pollutants, three with traffic-related pollutants, and three with traffic-related and other pollutants from industrial and filling station sources. The ISAAC questionnaire survey was given to all of the students except to those in the 1st grade who were presumed to be less likely to be exposed to the school environment than the remainder of the students attending those seven schools. The assessment of allergic disease was conducted on a total of 4,545 students. Three school zones with critical exposure were selected within each school and they were evaluated based on the levels of black carbon (BC), PM10, SO2, NO2, and O3. There was a significant increase in the risks based on the odds ratios of treatment experiences (within 1 year) for allergy-related diseases such as asthma and allergic rhinitis (a) in the school group with traffic-related pollutants and the school group with complex pollutants were 2.12 (1.41-3.19) and 1.59 (1.06-2.37), respectively, in comparison to the school groups with no exposure to pollutants. This was determined based on the odds ratio of symptoms and treatment experiences for allergy-related diseases by group based on the home town zone as a reference. Also, in the case of atopic dermatitis, the odds ratio of treatment experiences (within 1 year) was 1.42 (1.02-1.97), which indicated elevated risks compared to the students in the S1 school. A regression analysis was used to assess the relationship between the substances and the symptomatic experiences within the last year. There were significant increases in the odds ratio of the symptoms associated with allergic rhinitis and the BC and SO2 in the complex pollution areas. The results of the assessment of the relationship between atopic dermatitis-associated symptoms and O3 showed that the odds ratio increased with statistical significance.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a predominant amount of 3-hydroxyvalerate by engineered Escherichia coli expressing propionate-CoA transferase.

AIMS: Of the biodegradable polyhydroxyalkanoates (PHAs), poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is often considered for fabrication of biocompatible and absorbable medical devices and other applications. Depending on the application, however, specific mechanical or processing properties must be improved. To address these required properties, we sought to alter the monomer composition of the copolymer by a combination genetic engineering in an Escherichia coli host and carbon substrate feeding. METHODS AND RESULTS: We applied a new method of 3-hydroxyvalerate (3HV) monomer synthesis to produce a co-polymer by the introduction of a propionyl-CoA transferase gene (pct), along with PHA biosynthetic genes bktB, phaB and phaC from Ralstonia eutropha into engineered E. coli to produce P(HB-co-HV). The resulting strain successfully produced the copolymer containing an ultra-high 3HV monomer composition (over 80 wt%). CONCLUSIONS: To the best of our knowledge, the P(HB-co-HV) production strain constructed here synthesized polymer with the highest 3HV content of any engineered E. coli strain. This strain could also produce P(HB-co-HV) with the use of lower concentrations of propionate in the growth medium, compared to other reported strains, which could avoid the known growth inhibition from propionate in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyhydroxyalkanoates have been emphasized as a potential alternative for petroleum-based plastics by virtue of their physical properties and environmentally friendly characteristics. The copolymer produced in this work validates our genetic engineering approach and suggests that the Pct enzyme is a more efficient method for production of propionyl-CoA, the 3-hydroxyvaleryl-CoA precursor.

Clinical outcome of preemptive kidney transplantation in patients with diabetes mellitus.

End-stage renal disease (ESRD) caused by diabetic nephropathy is increasing throughout the world. The survival of diabetic patients treated by transplantation has improved nowadays. Although recent studies have demonstrated preemptive kidney transplantation to be associated with better graft survival in CKD patients, the effect of pre-transplantation dialysis on graft outcomes among diabetic ESRD patients is unclear. This analysis summarized our experience with preemptive kidney transplantation in diabetic ESRD patients by retrospectively comparing 70 such patients transplanted between 1995 and 2009. These 70 patients were divided into two groups: 30 patients underwent preemptive and the other 40 transplantation after maintenance hemodialysis or peritoneal dialysis. We compared graft survivals, acute rejection episodes, postoperative complications, and delayed graft function rates. The 10-year patient survival of 100% in the preemptive group was similar to that of the nonpreemptive group (85%, P = .11). But the 10 year graft survival was higher among the preemptive than the nonpreemptive group (100% vs 75%, P = .02). Pre-transplantation modality did not affect graft survival. Therefore, preemptive kidney transplantation should be applied to eligible patients with diabetic ESRD.